U.S. patent application number 09/909238 was filed with the patent office on 2004-12-02 for recombinant influenza viruses with bicistronic vrnas coding for two genes in tandem arrangement.
Invention is credited to Hobom, Gerd, Menke, Annette, Meyer-Rogge, Sabine.
Application Number | 20040241139 09/909238 |
Document ID | / |
Family ID | 33458029 |
Filed Date | 2004-12-02 |
United States Patent
Application |
20040241139 |
Kind Code |
A1 |
Hobom, Gerd ; et
al. |
December 2, 2004 |
Recombinant influenza viruses with bicistronic vRNAs coding for two
genes in tandem arrangement
Abstract
The invention relates to recombinant influenza viruses for
high-yield expression of incorporated foreign gene(s), which are
genetically stable in the absence of any helper virus and which
comprise at least one viral RNA segment being a tandem bicistronic
RNA molecule coding for two genes in tandem, in said tandem
bicistronic RNA molecule one of the standard viral genes being in
covalent junction with a foreign, recombinant gene and having an
upstream splice donor and a downstream splice acceptor signal
surrounding the proximal coding region. The invention further
provides a method for obtaining attenuated viruses which resist
reassortment dependent progeny production in case of chance
superinfections by wild-type influenza viruses; a method for the
production of said recombinant influenza viruses; pharmaceutical
compositions comprising said recombinant influenza viruses; and the
use of said recombinant influenza viruses for preparing medicaments
for vaccination purposes.
Inventors: |
Hobom, Gerd; (Giessen,
DE) ; Menke, Annette; (Marburg, DE) ;
Meyer-Rogge, Sabine; (Laubach-Munster, DE) |
Correspondence
Address: |
NORRIS, MCLAUGHLIN & MARCUS, PA
875 THIRD STREET
18TH FLOOR
NEW YORK
NY
10022
US
|
Family ID: |
33458029 |
Appl. No.: |
09/909238 |
Filed: |
July 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60220889 |
Jul 26, 2000 |
|
|
|
Current U.S.
Class: |
424/93.2 ;
435/235.1; 435/456; 514/44R |
Current CPC
Class: |
C12N 2840/20 20130101;
A61K 2039/5254 20130101; C07K 2319/00 20130101; A61K 48/00
20130101; C12N 15/86 20130101; C12N 2840/44 20130101; C07K 14/005
20130101; C12N 2760/16161 20130101; A61K 2039/5256 20130101; C12N
7/00 20130101; C12N 2760/16143 20130101; C12N 2760/16122
20130101 |
Class at
Publication: |
424/093.2 ;
514/044; 435/235.1; 435/456 |
International
Class: |
A61K 048/00; C12N
007/00; C12N 015/86 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 20, 2000 |
EP |
00115626.4 |
Claims
1. A recombinant influenza virus for high-yield expression of
incorporated foreign gene(s), which is genetically stable in the
absence of any helper virus and which comprises at least one viral
RNA segment being a bicistronic RNA molecule coding for two genes
in tandem arrangement (tandem RNA segment), in said tandem RNA
segment one of the standard viral genes being in covalent junction
with a foreign, recombinant gene and said tandem RNA segment having
an upstream splice donor and a downstream splice acceptor signal
surrounding the proximal coding region.
2. The recombinant influenza virus of claim 1, wherein the tandem
RNA segment contains one of the standard viral genes in distal mRNA
position behind a foreign, recombinant gene in proximal position,
or vice versa, both in antisense orientation with regard to the
viral RNA as present within the virus.
3. The recombinant influenza virus of claim 1 or 2, wherein at
least one of the regular viral RNA segments is replaced by a tandem
RNA segment, preferably the replaced regular viral RNA segment is
selected from the neuraminidase segment, hemaglutinin segment and
NS segment.
4. The recombinant influenza virus of claims 1 to 3, wherein the
splice donor and splice acceptor signals are selected from
sequences as present in influenza WSN segment 7 and 8 or other
partially effective splice reactin substrates.
5. The recombinant influenza virus of claim 4, wherein the splice
donor and splice acceptor signals are selected from sequences as
present in influenza WSN segment 7.
6. The recombinant influenza virus according to claims 1 to 5,
wherein one or more of the regular viral RNA segments, differing
from said at least one tandem RNA segment, comprises a vRNA
encoding a foreign gene which may or may not be in covalent
connection to one of the viral genes, and preferably one or more of
the regular viral RNA segments has (have) been deleted and replaced
by a tandem vRNA encoding in addition a foreign gene.
7. The recombinant influenza virus according to claims 1 to 6, in
which the terminal viral RNA sequences of one or more of the
regular segments and/or of the at least one tandem RNA segment,
which are active as the promoter signal, have been modified by
nucleotide substitutions in up to five positions, resulting in
improved transcription rates of both the vRNA promoter as well as
the cRNA promoter as present in the complementary sequence.
8. The recombinant influenza virus of claim 7, wherein the 12
nucleotide conserved influenza 3' terminal sequence has been
modified by replacement of one to three nucleotides occurring in
said sequence at positions 3, 5 and 8 relative to the 3' end by
other nucleotides, and/or wherein the 13 nucleotide conserved
influenza 5' terminal sequence has been modified by replacement of
one or two nucleotides occurring in said sequence at positions 3
and 8 by other nucleotides.
9. The recombinant influenza virus of claim 8, wherein the
replacements in the 3' terminal nucleotide sequence comprises the
modifications G3A and C8U.
10. The recombinant influenza virus of claim 9, wherein the
replacements in the 3' terminal nucleotide sequence comprises the
modifications G3A, U5C and C8U, or G3C, U5C and C8G.
11. The recombinant influenza virus of claim 10, which comprises a
3' terminal nucleotide sequence of (5')-CCUGUUUCUACU-3'.
12. The recombinant influenza virus according to claims 7 to 12,
wherein the 5' terminal nucleotide sequence comprises the
modifications U3A and A8U resulting in a 5'-terminal sequence of
5'-AGAAGAAUCAAGG.
13. The recombinant influenza virus according to claims 1 to 12,
which is a recombinant influenza A virus.
14. The recombinant influenza virus according to claims 1 to 13, in
which the foreign gene(s) in the tandem RNA segment code for
proteins and/or glycoproteins which are secreted from cells
infected with the recombinant virus.
15. The recombinant influenza virus according to claims 1 to 13, in
which the foreign gene(s) in the tandem RNA segment code for
proteins or artificial polypeptides designed to support an
efficient presentation of inherent epitopes at the surface of
infected cells, for stimulation of a B cell and/or T cell
response.
16. The recombinant influenza virus according to claims 1 to 13, in
which the foreign gene(s) in the tandem RNA segment is a nucleotide
sequence causing viral attenuation.
17. The recombinant influenza virus of claim 16, wherein the
foreign gene is coding for part of or for the entire viral
neuraminidase gene in antisense orientation.
18. The recombinant influenza virus of claim 17, wherein the
neuraminidase gene in antisense orientation is attached to the
hemaglutinin vRNA segment, and optionally another gene or reporter
gene is encoded in a second tandem vRNA, preferably in conjunction
with NS2.
19. A method for the production of recombinant influenza viruses as
defined in claims 1 to 18 comprising (a) RNA polymerase I synthesis
of recombinant vRNAs in vivo, in antisense or in sense tandem
design, (b) followed by infection with an influenza carrier strain
constructed to include flanking ribozyme target sequences in the
corresponding viral RNA segment, and (c) thereafter selective vRNA
inactivation through ribozyme cleavage.
20. A pharmaceutical composition comprising a recombinant influenza
virus according to claims 1 to 18, preferably a recombinant
influenza virus of claims 16 to 18.
21. Use of a recombinant influenza virus according to claims 1 to
18, preferably a recombinant influenza virus of claims 16 to 18,
for preparing a medicament for vaccination purposes.
22. The use according to claim 21, wherein the medicament (a) is
suitable against influenza and/or against other infections; (b) is
present in form of inactivated preparations; and/or (c) is present
in form of live recombinant viruses.
23. Use of a recombinant influenza virus according to claims 1 to
18 for preparing agents for somatic gene therapy.
24. Use of a recombinant influenza virus according to claims 1 to
18 for preparing agents, for transfer and expression of foreign
genes into cells infected by such viruses.
25. Use of a recombinant influenza virus according to claims 1 to
18 for preparing agents for transfer and expression of RNA
molecules into cells infected by such viruses.
26. The use of claim 24, wherein the RNA molecules to be expressed
are antisense sequences or double-strand sequences relative to the
target cell cellular mRNA molcules, and/or the agent is suitable
for sequence-specific gene silencing, preferably by antisense RNA
or RNA interference mechanisms.
27. The use according to claims 23 to 26, wherein the agents are
applicable in ex vivo and in vivo application schemes.
28. A method for the production of proteins or glycoproteins which
comprises utilizing a recombinant influenza virus according to
claims 1 to as expression vector.
29. The method of claim 28, wherein the production is performed in
cell culture cells or in fertilized chicken eggs.
30. A method for preventing and/or treating influenza which
comprises administering an effective amount of a recombinant
influenza virus according to claims 1 to 18, preferably of a
recombinant influenza virus according to claims 16 to 18, to the
mammal to be treated.
31. A method for somatic gene therapy, which method comprises
subjecting the organism to be treated with a recombinant influenza
virus according to claims 1 to 18.
32. A method for transfer and expression of foreign genes into
cells, and for transfer and expression of RNA molecules into cells,
which method comprises infecting the cells with a recombinant
influenza virus according to claims 1 to 18.
33. Use of a recombinant influenza virus according to claims 1 to
18 for preparing agents for immunotherapy, preferably for
autologous immunotherapy.
34. A method for an immunotherapy which comprises ex vivo infection
of immune cells, preferably dentritic cells, with a recombinant
influenza virus according to claims 1 to 18, and introduction of
the transduced cells into the patient.
35. A method for the induction of antibodies which comprises
utilizing a recombinant influenza virus according to claims 1 to 18
as an immunogen.
Description
FIELD OF THE INVENTION
[0001] The invention relates to recombinant influenza viruses for
high-yield expression of incorporated foreign gene(s), which are
genetically stable in the absence of any helper virus and which
comprise at least one viral RNA segment being a tandem bicistronic
RNA molecule coding for two genes in tandem, in said tandem
bicistronic RNA molecule one of the standard viral genes being in
covalent junction with a foreign, recombinant gene and having an
upstream splice donor and a downstream splice acceptor signal
surrounding the proximal coding region. In particular the above
tandem bicistronic RNA molecule contains one of the standard viral
genes in distal mRNA position behind a foreign, recombinant gene in
proximal position, or vice versa, both in antisense orientation
with regard to the viral RNA within the virus. For simultaneous
expression of both genes the proximal reading frame is flanked by
splice donor and acceptor signals which have the quality to allow a
partial yield of spliced mRNA only, i.e., resulting in the presence
of both, spliced and unspliced mRNA simultaneously.
[0002] The invention further provides a method for obtaining
attenuated viruses which resist reassortment dependent progeny
production in case of chance superinfections by wild-type influenza
viruses; a method for the production of said recombinant influenza
viruses; pharmaceutical compositions comprising said recombinant
influenza viruses; and the use of said recombinant influenza
viruses for preparing medicaments for vaccination purposes.
TECHNICAL BACKGROUND
[0003] Redesigning influenza virus into a vector system for
expression of foreign genes similar to what has been achieved in
several other thoroughly studied viruses such as adenovirus,
retrovirus, Semliki Forest virus or Rabies virus has the advantage
of an industrially well established mode of cheap propagation for
influenza in fertilized chicken eggs leading to rather high titers
(above 10.sup.10/ml). On the other hand none of the constituent
vRNA segments may be deleted from the influenza genome according to
our present knowledge, and give room for large-size foreign
insertions. Only small fragments of foreign polypeptide chains such
as B cell epitopes (10 to 15 amino acids) may be inserted into
selected positions within two of the viral proteins, i.e. in
exchange for one of the variable antigenic regions located at the
surface of hemagglutinin (Muster et al., Mucosal model of
immunization against human immunodeficiency virus type 1 with a
chimeric influenza virus, J. Virol. 69 (11), 6678-6686 (1995)), or
into the stalk sequence of viral neuraminidase (Garcia-Sastre and
Palese, The cytoplasmic tail of the neuraminidase protein of
influenza A virus does not play an important role in the packaging
of this protein into viral envelopes, Virus Res. 37, 37-47 (1995)),
and be stably maintained as functional fusion proteins. Constructs
of this kind turned out to be useful for experimental vaccination
in a few cases studied, but only rather few clearly defined epitope
sequences (of ten to twelve amino acids each) are known today, and
some of them might also be misfolded within such restricted fusion
protein positions, or in other cases interfere with formation of
the correct tertiary structure and function of their host
polypeptide chains.
[0004] Incorporation of a full-size foreign protein into influenza
virus via reverse genetics, encoded by an independent ninth vRNA
molecule in addition to its regular set of eight standard vRNA
segments is without special provisions only transiently possible
(Luytjes et al., Amplification, expression, and packaging of a
foreign gene by influenza virus. Cell 59, 1107-1113 (1989); Enami
et al., An influenza virus containing nine different RNA segments,
Virology 185, 291-298 (1991)). In the absence of a continuous
selective pressure any additional recombinant vRNA segment cannot
be stably maintained as long as the wildtype promoter sequence is
used on that ninth vRNA segment, and it will inadvertently be lost
after few steps of viral propagation.
[0005] Using a different system of influenza reverse genetics
developed in our laboratory (Zobel et al., RNA polymerase I
catalysed transcription of insert viral cDNA, Nucleic Acids Res.
21, 3607-3614 (1993); Neumann et al., RNA polymerase I-mediated
expression of influenza viral RNA molecules, Virology 202, 477-479
(1994)), which was built around in vivo synthesis of recombinant
vRNA molecules by cellular RNA polymerase I transcription of the
respective template cDNA constructs, modified terminal viral RNA
sequences (hereinafter "promoter-up mutations" or promoter-up
variants") have been designed by nucleotide substitutions (Neumann
and Hobom, Mutational analysis of influenza virus promoter elements
in vivo, J. Gen. Virol. 76, 1709-1717 (1995); WO 96/10641). The
above promoter-up variants carry up to five nucleotide
substitutions (in promoter-up variant 1920; see Flick and Hobom, j.
Gen. Virol. 80, 2565-2572 (1999)). When these promoter-up variants
are attached to a recombinant ninth vRNA segment its increased
transcription and amplification rate will not only compensate for
the losses suffered spontaneously, but even cause accumulation of
the foreign vRNA segment during simple viral passaging, in the
absence of any selection.
[0006] However, due to its over-replication relative to all of the
regular influenza vRNA segments (which of course are connected to
wild-type promoter sequences) after catching up with the others the
foreign segment will become over-abundant. This increasingly will
result in viral particles that have incorporated several copies of
recombinant vRNA, but no longer have a full set of all eight
standard segments incorporated among an average of about 12-15 vRNA
molecules present within a virion. Such particles are defective and
will not result in plaque formation, hence after an initial
increase of recombinant viral particles during the first steps of
propagation a dramatic decrease is observed, usually at the third
or fourth step of viral passaging, depending on the size of the
recombinant vRNA and the level of the promoter-up mutation
attached.
[0007] A balanced situation with regard to the insert length and
the level of promoter activity can be achieved, and has been
propagated in a particular case over 11 passages, with essentially
stable levels of recombinant viruses among a majority of helper
viruses (around 80%) during these steps. If a full-level
promoter-up mutation is used (1104 or the variant 1920, see below)
a balanced-level propagation is reached in conjunction with a
recombinant vRNA size of 4000 nucleotides (Maysa Azzeh, Ph.D.
Thesis, Univ. Giessen (2000)).
[0008] In all of these preparations, both in transiently achieved
increased yields (Up to 40% of recombinants after three or four
steps of viral passage), and in a balanced propagation of
recombinant influenza viruses (10-20%) the respective viral progeny
inadvertently constitute mixtures with a majority of
non-recombinant helper viruses. These result both from a
statistical mode of packaging vRNA molecules into a virion (the
ninth segment may not be co-packaged), and from the fraction of
cells solely infected by helper virus.
[0009] To solve the problems of fractional yields and of
instability during viral propagation of recombinant influenza, it
was suggested to use a recombinant influenza virus for high-yield
expression of incorporated foreign gene(s), which is genetically
stable in the absence of any helper virus and which comprises at
least one viral RNA segment being an ambisense RNA molecule
(designated "ambisense RNA segment") and containing one of the
standard viral genes in sense orientation and a foreign,
recombinant gene in anti-sense orientation, or vice versa, in
overall convergent arrangement (PCT/EP00/01903). The ambisense RNA
segment preferably should contain the promoter-up mutations. The
PCT/EP00/01903 moreover discloses a method of constructing specific
influenza carrier (helper) strains carrying one or more ribozyme
target sites (of type one) in vRNA flanking positions
comprising
[0010] (a) RNA polymerase I synthesis of recombinant vRNAs in vivo,
carrying two different 3' promoter sequences in tandem (an external
promoter-up variant and an internal wild-type promoter), which are
separated by a second type of ribozyme target sequence, and which
carry the said internal ribozyme target sites of type one;
[0011] (b) followed by infection of an influenza wildtype
strain;
[0012] (c) thereafter amplification through simple steps of viral
propagation; and
[0013] (d) finally isolation through removal of their external 3'
promoter sequence by ribozyme cleavage through infection of cells
expressing ribozyme type 2, followed by plaque purification.
[0014] The resulting special helper virus strains carrying a vRNA
segment with external ribozyme target sites of type 1 in exchange
for the equivalent regular vRNA molecule are then used for the
rescue of ambisense RNA molecules. These are exclusively maintained
in the recombinant viruses after passage of viral propagation
through ribozyme (type 1) containing host cells, which will destroy
the sensitive vRNA molecules of the specially prepared helper
viruses.
[0015] However, the above ambisense constructs are susceptible to
(intra-nuclear) mRNA double-strand formation, which will partially
reduce the expression rates of both the ambisense genes, in
particular the gene driven by the (weaker) cRNA promoter. The
fluctuating extent of this effect made it difficult to bring the
expression rate of the influenza gene within the ambisense segment
into balance with other influenza genes. This was the problem to be
solved with the present invention.
SUMMARY OF THE INVENTION
[0016] Starting out from two observations in this laboratory which
are discussed above and which concern two hitherto unsuspected
properties of influenza viral RNA polymerase in its interaction
with terminally adapted influenza-specific RNA molecules, stable
recombinant influenza viruses were found, which solve the above
problems.
[0017] The recombinant viruses of the present invention can be used
for cheap propagation in fertilized eggs, either for production of
those recombinant viruses themselves or for production of foreign
proteins or glycoproteins encoded by them, and hence find
application in (glyco)protein production or in providing vector
systems for somatic gene therapy or in being used as vaccination
agents.
[0018] Thus, the present invention provides
[0019] (1) a recombinant influenza virus for high-yield expression
of incorporated foreign gene(s), which is genetically stable in the
absence of any helper virus and which comprises at least one viral
RNA segment being a bicistronic RNA molecule coding for two genes
in tandem arrangement (hereinafter "tandem bicistronic RNA segment"
or "tandem RNA segment"), in said tandem RNA segment one of the
standard viral genes being in covalent junction with a foreign,
recombinant gene and said tandem RNA segment having an upstream
splice donor and a downstream splice acceptor signal surrounding
the proximal coding region;
[0020] (2) a preferred embodiment of the recombinant influenza
virus defined in (1) above, in which the terminal viral RNA
sequences of said at least one tandem RNA segment, which are active
as the promoter signal, have been modified by nucleotide
substitutions in up to five positions, resulting in improved
transcription rates of both the vRNA promoter as well as the cRNA
promoter as present in the complementary sequence;
[0021] (3) a method for the production of recombinant influenza
viruses as defined in (1) and (2) above comprising
[0022] (a) RNA polymerase I synthesis of recombinant vRNAs in vivo,
in antisense, or in sense tandem design,
[0023] (b) followed by infection with an influenza carrier strain
constructed to include flanking ribozyme target sequences in the
corresponding viral RNA segment, i.e., coding for the same viral
gene as present in the tandem segment distal position, and
[0024] (c) thereafter selective vRNA inactivation through ribozyme
cleavage;
[0025] (4) a pharmaceutical composition comprising a recombinant
influenza virus as defined in (1) and (2) above;
[0026] (5) the use of a recombinant influenza virus as defined in
(1) and (2) above for preparing a medicament for vaccination
purposes;
[0027] (6) the use of a recombinant influenza virus as defined in
(1) and (2) above for preparing agents for somatic gene
therapy;
[0028] (7) the use of a recombinant influenza virus as defined in
(1) and (2) above for preparing agents for transfer and expression
of foreign genes into cells (abortively) infected by such
viruses;
[0029] (8) the use of a recombinant influenza virus as defined in
(1) and (2) above for preparing agents for transfer and expression
of RNA molecules into cells infected by such viruses;
[0030] (9) a method for the production of proteins or glycoproteins
which comprises utilizing a recombinant influenza virus as defined
in (1) and (2) above as expression vector;
[0031] (10) a method for preventing and/or treating influenza which
comprises administering a recombinant influenza virus as defined in
(1) and (2) above to the mammal to be treated, i.e., a vaccination
method utilizing said recombinant virus;
[0032] (11) a method for somatic gene therapy, which method
comprises subjecting the organism to be treated with a recombinant
influenza virus as defined in (1) and (2) above;
[0033] (12) a method for transfer and expression of foreign genes
into cells, and for transfer and expression of RNA molecules into
cells, which method comprises infecting the cells with a
recombinant influenza virus as defined in (1) and (2) above;
[0034] (13) use of a recombinant influenza virus as defined in (1)
and (2) above for preparing agents for autologous
immunotherapy;
[0035] (14) a method for an immunotherapy which comprises ex vivo
infection of immune cells with a recombinant influenza virus as
defined in (1) and (2) above, and introduction of the transduced
cells into the patient; and
[0036] (15) a method for the induction of antibodies which
comprises utilizing a recombinant influenza virus as defined in (1)
and (2) above as an immunogen.
[0037] The invention is described in more detail below.
BRIEF DESCRIPTION OF THE FIGURES
[0038] FIG. 1 shows the basepair substitution analysis according to
the vRNA `corkscrew` structure:
[0039] (A) `Corkscrew` conformation of the vRNA promoter drawn
against a schematic indication of interacting tripartite viral
polymerase. Paired positions exchanged in individual experiments
are indicated by numbers, nucleotides {overscore (3)} or {overscore
(8)} are counted from the 3' end. pHL2024 containing promoter-up
mutation `1104` is used as the reference construct (=100%) in all
of the CAT assays, while pHL2428 represents the wild-type promoter
structure.
[0040] (B) CAT analysis of a series of substitution variants in
positions 3 and 8 from the 5' end as indicated above the lanes; 50
.mu.l of cell lysate obtained from 10.sup.6 MDCK cells infected in
the first viral passage with recombinant viral progeny.
[0041] (C) pHL2024 and pHL1920 comparative CAT analysis, in 100
fold dilution relative to (B), i.e., obtained from 0.5 .mu.l of
cell lysate in 3 h reaction time.
[0042] FIG. 2: Vector plasmid pHL1920, the excact sequence of the
3888 bps circular DNA is shown in SEQ ID NO: 20
[0043] FIG. 3 shows the genetic structure and the RNA transcription
products of influenza model tandem expression constructs. Heavy
lines for the plasmid cDNA constructs refer to double-stranded DNA,
while single-stranded RNA molecules are represented by thin lines,
and their 5' to 3' directionalities are marked by arrows. Standard
modifications at their 5' and 3' ends are indicated by a dot (5'
cap structure) and A.sub.n (3' poly-adenylation), both are absent
in the primary anti-sense transcription product, the viral RNA
(vRNA), which is transcribed by cellular RNA polymerase I (RPoI).
Full-length mRNA, is synthesized by influenza viral polymerase
(virPo), and a partial splice reation results in a functional yield
of shorter mRNA.sub.2 molecules. While both of the reporter genes
are indicated on the DNA level, together with the positions of
splice donor (D) and acceptor (A) signal sequences as well as the
promotor (p.sub.I) and terminator (t.sub.I) elements for RNA
polymerase I start and stop, on the RNA level only those genes and
splice signals are marked that are actually translated into protein
or actively involved in splicing. The
chloramphenicol-acetyltransferase gene (CAT) has been inserted in
proximal position in pHL3196 and pHL3235, and in distal position in
pHL3224 and pHL3236 (see FIGS. 5 to 8), while green fluorescent
protein (GFP) in each case is located in alternate location. All
vRNA molecules--and hence, also the cDNA constructs--carry sequence
variations at their 3' ends, which together constitute the 1104
promoter-up mutations: G3A, U5C, C8U (nucleotide positions counted
from the 3' vRNA end). pHL3235 and pHL3236 vRNAs are extended in
size by about 1000 nucleotides of untranslated sequence relative to
pHL3196 and pHL3226: 2600 instead of 1600 nucleotides in lengths.
For full-size representation of circular plasmid DNAs see FIGS.
5-8, for CAT expression data of all infected by recombinant
influenza viruses carrying the respective viral RNAs see FIG.
4.
[0044] FIG. 4 shows the CAT assay results for the group of tandem
vRNA plasmid constructs as described in the Example. In particular,
the ratio between chloramphenicol (bottom line) and
acetylchloramphenicol (upper three lines) in a flash-CAT assay,
after the 2.sup.nd (A) and 4.sup.th (B) passage of recombinant
viruses carrying the reportergene CAT, can be determined from said
figure. The following constructs were utilized:
[0045] pHL1844 (control): monocistronic CAT-construct downstream of
promoter variant 1104.
[0046] pHL3196: tandem construct, p-CAT-GFP resulting in a vRNA
having a total length of 1530 nucleotides (not "extended"), see
also FIG. 5.
[0047] pHL3235: tandem construct, p-CAT-GFP resulting in a vRNA
having a total length of 2550 nucleotides ("extended"), see also
FIG. 7.
[0048] pHL3224: tandem construct, p-CAT-GFP resulting in a vRNA
having a total length of 1700 nucleotides (not "extended"), see
also FIG. 6.
[0049] pHL3236: tandem construct, p-CAT-GFP resulting in a vRNA
having a total length of 2720 nucleotides ("extended"), see also
FIG. 8.
[0050] pHL2899: ambisense construct, p.sub.v-CAT.fwdarw.
.rarw.GFP-p.sub.c resulting in an RNA having a total length of 1500
nucleotides.
[0051] pHL2960: ambisense construct, p.sub.v-CAT.fwdarw.
.rarw.GFP-p.sub.c resulting in an RNA having a total length of 1500
nucleotides.
[0052] The five constructs on the left side were transfected into
the cell DNA without the use of "booster" plasmides, the four
constructs on the right side were, however, transfected with the
"booster" plasmides, which gives a jump-start of the constructs due
to recombinant vRNA amplification prior to helper virus injection,
equivalent to an advantage of about two passages. The "booster"
plasmides comprise expression constructs for the nucleoprotein as
well as the three subunits of influenza viral polymerase, each
downstream of an RNA polymerase II promoter and in an mRNA forming
cassette.
[0053] While the ambisense construct having the CAT-reporter gene
in the weaker position, i.e. behind the cRNA promoter (pHL2899), is
only expressed moderately, this is not the case in the respective
tandem construct having the CAT-reporter gene in the weaker
position, viz. pHL3224 or pHL3236. Further, the "extension" of the
vRNA by 1020 non-translated nucleotides (at the 3' end) is
tolerated without significant decrease of expression (see pHL3235
versus pHL3196).
[0054] FIG. 5: Vector plasmid pHL3196, the exact sequence of the
4500 bps circular DNA is shown in SEQ ID NO:21.
[0055] FIG. 6: Vector plasmid pHL3224, the exact sequence of the
4721 bps circular DNA is shown in SEQ ID NO:22.
[0056] FIG. 7: Vector plasmid pHL3235, the exact sequence of the
5517 bps circular DNA is shown in SEQ ID NO:23.
[0057] FIG. 8: Vector plasmid pHL3236, the exact sequence of the
5699 bps circular DNA is shown in SEQ ID NO:24.
DETAILED DESCRIPTION OF THE INVENTION
[0058] According to the present invention "influenza virus"
embraces influenza A virus, influenza B virus and influenza C
virus, with influenza A virus being preferred.
[0059] "Bicistronic" according to the present invention refers to a
viral RNA segment, vRNA, cRNA or mRNA that includes two independent
genes in covalent junction; in a preferred version one of these
genes is of viral origin, while the other one codes for a foreign,
recombinant gene product.
[0060] "Proximal" and "proximal position" according to the present
invention refers to the 5' portion of one of the genes in the
bicistronic viral mRNA, i.e., ahead (upstream) of the second gene
in "distal position".
[0061] A "mammal" according to the present invention includes
humans and animals. "Organism" embraces prokaryotic and eukaryotic
systems as well as multicellular systems such as vertebrates
(including mammals) and invertebrates.
[0062] "Infected cells" and "infecting cells" according to the
present invention also include "abortively infected cells" and
"abortively infecting cells", respectively.
[0063] In a preferred influenza virus according to embodiment (1)
at least one of the regular viral RNA segments is replaced by a
tandem RNA segment which contains one of the standard viral genes
in distal position, and a foreign, recombinant gene in proximal
position, both in anti-sense orientation, or vice-versa. It is
moreover preferred that in the tandem RNA molecule said foreign
recombinant gene is covalently bound to one of the viral genes
while the original vRNA segment coding for the same gene is deleted
from the recombinant virus by specific ribozyme cleavage.
[0064] The foreign gene(s) in tandem covalent junction with the
viral gene(s) preferably code for proteins and/or glycoproteins
which are secreted from cells infected with the recombinant virus,
such as lymphokines, or code for glycoproteins that are
incorporated into the virion as well as the plasma membrane of the
infected cell. In another preferred embodiment the foreign gene(s)
in tandem covalent junction with the viral gene(s) code for
proteins or artificial polypeptides designed to support an
efficient presentation of inherent epitopes at the surface of
infected cells, for stimulation of B cell and/or T cell response.
Such proteins or artificial polypeptides constitute for instance a
tumor antigen or an artificial oligomeric series of T cell epitopes
that have been identified within a polypeptide chain. Finally, the
foreign gene(s) may be suitable for transfer and expression of RNA
molecules, including antisense RNAs and ribozymes, into cells. Such
recombinant influenza viruses are suitable for sequence specific
gene silencing, for example by antisense or RNA interference
mechanisms.
[0065] A preferred recombinant virus of the invention is where in
the regular viral RNA segments one or both of the standard
glycoproteins hemagglutinin and neuraminidase have been exchanged,
preferably into fusion glycoproteins consisting of an anchor
segment derived from hemagglutinin and an ectodomain obtained from
the foreign source, viral or cellular, or in which such recombinant
glycoprotein has been inserted as a third molecular species in
addition to the remaining standard components.
[0066] As set forth in embodiment (2) above, a preferred
recombinant virus of the invention is where the terminal viral RNA
sequences, which are active as promoter signal, have been modified
by nucleotide substitution in up to 5 positions, resulting in
improved transcription rates (of both the vRNA promoter and in the
cRNA promoter as present in the complentary sequence) as well as
enhanced replication and/or expression rates relative to the
wild-type sequence. Said modified terminal viral RNA sequences
differ from the wild-type sequence in that in said tandem vRNA
segment the 12 nucleotide conserved influenza 3' terminal sequence
has been modified by replacement of one to three nucleotides
occurring in said sequence at positions 3, 5 and 8 relative to the
3' end by other nucleotides provided that the nucleotides
introduced in positions 3 and 8 are forming a base pair (i.e., if
the nucleotide position 3 is G, than that in position 8 is C; if
the nucleotide in position 3 is C, than that in position 8 is G;
etc.).
[0067] The 3' conserved regions of the wild-type influenza virus
have the following sequences:
1 Influenza A: (5')-CCUGCUUUUGCU-3' Influenza B:
(5')-NN(C/U)GCUUCUGCU-3' Influenza C: (5')-CCUGCUUCUGCU-3'.
[0068] Moreover, the 13 nucleotide conserved influenza 5'-terminal
sequence may be modified by replacement of one or two nucleotides
occurring in said sequence as positions 3 and 8 by other
nucleotides, again provided that the introduced nucleotides are
forming a base pair. The 5' conserved regions of the wild-type
influenza virus have the following sequences:
2 Influenza A: 5'-AGUAGAAACAAGG Influenza B:
5'-AGUAG(A/U)AACA(A/G)NN Influenza C: 5'-AGCAGUAGCAAG(G/A):
[0069] Preferred influenza viruses of the invention are those
wherein in the 3' conserved region the replacements G3A and C8U
have been performed, more preferred are those where also the
replacement U5C has been performed (the above mutations are
annotated relative to the 3' end; such counting from the 3' end is
also indicated by a line on top of the digit, e.g., G {overscore
(3)}A). Another preferred influenza virus mutant comprises the
3'-terminal nucleotide sequence G3C, U5C and C8G (relative to the
3' end) resulting in the following 3' terminal nucleotide sequence
(5')-CCUGGUUCUCCU-3'. Among the influenza viruses defined
hereinbefore those having a 3'-terminal nucleotide sequence of
(5')-CCUGUUUCUACU-3' are most preferred. In case of an influenza A
virus the segment may further have the modifications U3A and A8U in
its 5' terminal sequence, in case of influenza C it may have the
modifications C3U and G8A in its 5' terminal sequence. The most
preferred influenza viruses of the present invention comprise the
following general structures:
3 Influenza A (mutant pHL1104): 5'-AGUAGAAACAGGNNNU.sub.5-6-
..(880-2300 ntds)..N'N'N'CC UGUUUCU-3' Influenza A (mutant
pHL1920): 5'-AGAGAACAGGNNNU.sub.5-6..(880-2300 ntds)..N'N'N'CC
UGUUUCU-3' Influenza A (mutant pHL1948):
5'-AGUAGAAACAAGGNNNU.sub.5-6..(880-2300 ntds)..N'N'N'CC UGUUUCU-3'
Influenza B: 5'-AGUAG(A/U)AAC(A/G)NNNNNU.sub.5-6..(880-2300 ntds)..
N'N'N'N'N'(C/U)GUUCUCU-3' Influenza C:
5'-AGAGUACAG(G/A)GU.sub.5-6..(880-2300 ntds)..CCCCUG UUCUCU-3'
[0070] In the above structures the variables are defined as
follows:
[0071] (1) Underlined and enlarged letters show the required
mutations relative to the wild-type sequence for preparing a
promoter mutant with enhanced properties;
[0072] (2) enlarged A in position 10 in the 5'-part of the
sequence: unpaired A residue, bulge-forming;
[0073] (3) (A/G) in one position: different isolates or single
segments with variable sequence at the respective position, which
are functionally interchangeable;
[0074] (4) N and N': positions undefined, but base-paired relative
to each other because of complementarity between the 5' and 3'
termini, different among the 8 segments, but constant for each
segment throughout all viral isolates;
[0075] (5) (880-2300 ntds): the lengths of the viral RNA segments,
in case of segments with foreign genes increased up to 4,000
nucleotides.
[0076] According to embodiments (1) to (3) the invention
provides
[0077] a stable recombinant influenza virus containing (up to)
seven regular vRNA segments plus one (or more) additional
bicistronic segment(s) coding for a foreign gene in covalent
conjunction with one of the influenza genes, in tandem arrangement,
and
[0078] a method for the construction of stable recombinant
influenza viruses through tandem arrangement of bicistronic vRNA
segments, which is also applicable as a method for attenuation and
for prevention of reassortment between co-infecting influenza
viruses.
[0079] Expression of both gene products in these constructions is
made possible by way of an upstream splice donor and a downstream
splice acceptor signal surrounding the proximal coding region of
such a quality that splicing does occur in part of the mRNA
molecules only, i.e., both mRNAs spliced and unspliced are present
in the infected cell. For compensation with regard to the vRNA
length the bicistronic segment is connected to a promoter variant
of enhanced replication and transcription rates as defined herein
before.
[0080] The splice donor and the splice acceptor signals are
selected from authentic sequences as present in influenza segments
7 and 8 or other partially effective splice reaction substrates,
preferably those of influenza virus WSN segment 7, i.e.,
5'-AG.sup..dwnarw.GTACGTTC-3' (donor) and
5'-GCTGAAAAATGATCTTCTTGAAAATTGCAG.sup..dwnarw.GC-3' (acceptor).
[0081] In a particular application of embodiments (1) to (3) the
tandem bicistronic mRNA codes for one of the viral genes, such as
hemagglutinin, in conjunction with all or part of the viral
neuraminidase coding sequence, in antisense orientation, while the
authentic neuraminidase vRNA segment is missing in these
recombinant viruses. In another variation of these constructs an
anti-neuraminidase ribozyme sequence is also provided together with
the (partial) neuraminidase antisense sequence, in the proximal
position of these bicistronic recombinant segments. Recombinant
viruses of this character are propagated in culture media with
addition of exogenous neuraminidase.
[0082] The absence of a functional neuraminidase gene serves as a
strong attenuation mechanism resulting in single-step infections of
such recombinant viruses only. While a functional neuraminidase
gene could be provided through another (wildtype) influenza virus
superinfecting the same cell, expression of that gene is very much
reduced through antisense RNA interaction and/or destruction of the
corresponding vRNA through ribozyme cleavage, designed to interfere
with production of infectious progeny even from co-infected cells;
as a barrier against reassortment in double infected cells.
[0083] Recombinant viral RNAs coding simultaneously for two genes
in tandem in a construct in which one of the viral genes is in
covalent junction with a foreign coding sequence, are constructed
via E. coli plasmid vector DNAs designed for an in vivo
transcription of minus-strand vRNAs by cellular RNA polymerase I.
In these constructs the gene in plus-strand proximal (upstream)
position is surrounded by splice signals of limited activity such
that both mRNAs, spliced and unspliced are present in the infected
cell. Either the foreign gene or the viral gene may be in that
upstream position. In the majority of applications the higher rates
of expression will be reserved for the foreign coding sequence,
while the lower expression rate of the viral gene is adapted to be
approximately in balance with expression of the other viral genes
encoded by the regular viral segments.
[0084] To achieve such a balanced rate of expression, the splice
signals and the promoter have to be chosen properly (Flick and
Hobom, Interaction of influenza virus, polymerase with viral RNA in
the `corkscrew` conformation, J. Gen. Virol. 80, 2565-2572 (1999)).
At an increased overall transcription rate; the resulting mRNAs
shall be spliced inefficiently if the viral gene is in the distal
(downstream) position. Vice-versa, if the foreign gene is in the
distal position, splicing to obtain the foreign mRNA shall be
achieved efficiently. Both designs serve to reach an
over-expression of the foreign gene relative to the viral gene, of
which the expression shall be in balance with the expression of the
other viral genes. Further, the promoter variant attached to the
bicistronic segment has the function to compensate for the
increased gene length by way of an increased replication rate.
[0085] The influenza vRNA segments preferably used for construction
of bicistronic segments include the neuraminidase (No. 6),
hemagglutinin (No. 4) and NS segment (No. 8). In the NS segment the
foreign gene may also substitute for the NS1 gene leaving the viral
NS2 gene in its place. These recombinant viruses can, as an
example, be made by the following procedure: A recombinant virus
population can be selected by repeated ribozyme-mediated cleavage
of helper-virus segments carrying ribozyme cleavage sites that
flank the same viral gene in the monocistronic segment as is
present in the bicistronic construct (PCT/EP00/01903). By serial
viral passaging and relying on the ouptut of reporter genes in
equivalently constructed bicistronic segments, a balanced mode of
expression can be achieved in choosing the right set of elements:
promoter, splice signals plus a limited variation in segment
length. The construct that gives rise to the balanced, stable
expression is then used as a basis for a multiple cDNA transfection
procedure in a helper-virus free design according to Neumann et
al., Proc. Natl. Acad. Sci. USA, Vol 96, 9345-9350 (August 1999).
The resulting recombinant influenza virus, obtained via single
plaques in pure helper-free state is subjected to another series of
propagation steps to finally evaluate its properties.
[0086] In a particular application this design is used for a
controlled mode of viral attenuation. Attenuation of influenza
viruses so far has been achieved in cold-sensitive mutants (Edwards
et al., J. Infect. Dis. 169, 68-76(1994)), by deletion of the NS1
gene (partial attenuation, Egorov et al., J. Virol. 72, 6437-6441
(August 1998) and Palese et al., Proc. Natl. Acad. Sci USA,
4309-4314 (April 2000)), or through deletion of the neuraminidase
gene (full attenuation, Kawaoka et al., J. Virol. 74, 5206-5212
(June 2000)). The latter approach is adapted here using a novel
technique for the attenuation, which for the first time is also
able to interfere with (chance) superinfection by wild-type
viruses.
[0087] In this embodiment of the invention a bicistronic cDNA
construct is achieved, which instead of a foreign gene is coding
either for part of or for the entire viral neuraminidase gene in
antisense orientation, with or without being surrounded both by
splice donor and acceptor elements. In another version of that
design a 2.times.50 nucleotide antisense segment complementary to
the 5'-terminal neuraminidase sequence has been cloned in flanking
positions relative to a ribozyme construct according to the
hammerhead design and oriented against a common GUC triplett within
the neuraminidase sequence. In a preferred design this antisense
expression construct has been attached to the hemagglutinin vRNA
segment, while another gene or reporter gene is encoded in a second
bicistronic vRNA, in conjunction with NS2.
[0088] Propagation of recombinant viruses deleted for the
neuraminidase (NA) gene requires an addition of external
neuraminidase to the medium. In the absence of neuraminidase,
infection by the NA deletion viruses is abortive: no infectious
progeny is produced. Upon co-infection (3:3) of recombinant viruses
together with wildtype viruses no progeny virus or plaque is
observed, which is attributed to antisense-blocked expression or
(partial) destruction of the neuraminidase segment originating from
the wild-type virus. Therefore, the recombinant viruses described
are not only attenuated in single infections, but simultaneously
interfere with wildtype virus superinfection, and therefore, no
re-assortment between the two viruses will occur.
[0089] The pharmaceutical composition according to embodiment (4)
above and the medicament of embodiment (5) above contain the
recombinant influenza virus in a pharmaceutically effective amount.
Besides said recombinant influenza virus, the pharmaceutical
composition and the medicament may contain further pharmaceutically
acceptable carrier substances well-known to a person skilled in the
art, such as binders, desintegrants, diluents, buffers,
preservatives, etc. The pharmaceutical composition and medicaments
are solid or liquid preparations and are suitable to be
administered orally, intravenously or subcutaneously.
[0090] The medicament according to embodiment (5) above is
preferably suitable as a medicament against influenza and/or
against other infections. The recombinant influenza virus may be
present in form of inactivated preparations or may be present in
form of live recombinant viruses, preferably as attenuated
viruses.
[0091] Live recombinant viral vaccines, live but attenuated
recombinant viral vaccines or inactivated recombinant viral vaccine
can be formulated. Inactivated vaccines are "dead" in the sense
that their infectivity has been destroyed. Ideally, the infectivity
is destroyed without affecting its immunogenicity. To prepare
inactivated vaccines, the recombinant virus may be grown in cell
cultures or in embryonated chicken eggs, purified, and inactivated
by formaldehyde or .beta.-propiolactone. The resulting vaccine is
usually administered intramuscularly.
[0092] Inactivated viruses may be formulated with suitable
adjuvants to enhance the immunological response. Such adjuvants
include, but are not limited to, mineral gels, e.g., aluminum
hydroxide, surface-active substances such as pluronic polyols,
lysolecithin, peptides, oil emulsions, and potentially useful human
adjuvants such as BCG.
[0093] Many methods may be used to introduce the vaccine
formulations above, for example the oral, intradermal,
intramuscular, intraperitoneal, subcutaneous, or intranasal routes.
Where a live recombinant virus vaccine is used, it is preferred to
introduce the formulation via the natural route of infection for
influenza virus.
[0094] The medicament according to embodiment (5) above is
preferably suitable for prophylactic or therapeutic vaccination, or
both, against influenza and other infections. For example,
recombinant viruses can be made for use in vaccines against HIV,
hepatitis B virus, hepatitis C virus, herpes viruses, papilloma
viruses, to name but a few. In one embodiment the recombinant virus
contains the genes for surface proteins of the viruses, in another
the genes for non-structural or regulatory genes. The recombinant
viruses may be present in form of inactivated preparations or may
be present in form of live recombinant viruses, or as live, but
attenuated viruses. In an attenuated virus the recombinant virus
would go through a single or at most very few propagation cycle(s)
and induce a sufficient level of immune response, but would not
cause disease. Such viruses lack one of the essential influenza
genes or contain mutations to introduce temperature
sensitivity.
[0095] The agents of embodiments (6)-(8) of the invention are
applicable in ex vivo and in vivo application schemes. The RNA
molecule to be expressed by means of the agent of the embodiment
(8) is of an antisense sequence or double strand sequence (in
ambisense bidirectional transcription) relative to a target
cellular mRNA molecule. In embodiment (8) the agent is preferably
suitable for sequence-specific gene silencing, preferably by
antisense RNA or RNA interference mechanisms.
[0096] The method for the production of proteins or glycoproteins
is preferably performed in cell culture cells or in fertilized
chicken cells in accordance with standard techniques within the
general knowledge of a person skilled in the art. The proteins or
glycoproteins to be expressed are those incorporated into the
ambisense construct as defined hereinbefore.
[0097] The methods according to embodiments (9) to (12), (14) and
(15) of the invention include the administration of an effective
amount to the mammal or the administration of a sufficient
infective dose of the recombinant virus to the cell system that is
used for ex vivo therapy or for in vitro investigations, whereby
the amount and dose will be determined by a person skilled in the
respective arts or knowledgeable of the desired treatments.
[0098] The agent of embodiment (14) of the invention is preferably
utilized to infect, transfect or transduce patient-derived immune
cells. The agent is suitable for treatment of cancer or chronic
viral infections. For this purpose, patient derived immune cells,
preferably dendritic cells, are ex vivo infected with recombinant
influenza viruses expressing, e.g., tumor antigens or viral
antigens. The transduced cells are then reintroduced into the
patient.
[0099] The preferred method for immunotherapy of embodiment (14) of
the invention is an autologous immunotherapy, wherein the cells
which are ex vivo infected are patient-derived and the transduced
cells are reintroduced into the patient. The diseases to be treated
by this method include cancer and chronic viral infections. For
details regarding such treatment see discussion of embodiment (13)
above.
[0100] The method for inducing antibodies according to embodiment
(15) of the invention is suitable for inducing antibodies to
foreign proteins including glycoproteins, following the
administration of protein or glycoprotein antigens as part of a
recombinant influenza virus in an authentic, conformation, whereby
the virus is purified by gentle procedures based on
hemagglutination, and the gene is expressed at high rates in the
infected cells.
[0101] As influenza viruses have a wide host range, recombinant
influenza viruses can be used to obtain strong immune responses in,
and isolate antibodies from, a wide range of animals, including,
but not limited to, fowl, pigs, horses, and mice. Further,
influenza viruses adapted to the mouse can be used for the
infection of mice by several routes including the intranasal route.
This results in infection of the pharyngeal mucosal cells and
results in an additional type of B cell response (e.g., as
recognized in the ratio of IgG to IgA). Mice are of particular
utility in the induction of immune responses in transgenic mice
that have been engineered to express human antibodies. As gentle
procedures based on hemadsorption are used to purify influenza
viruses, antibodies to antigens in native conformation can be
isolated from the infected mammals. The preset invention further
illustrated by the following, non-limiting Example:
EXAMPLE
[0102] Model tandem bicistronic expression constructs using
reporter genes CAT and GFP.
[0103] Objective: Measurements of relative expression rates for CAT
in proximal and distal position, with live observation of GFP
fluorescence in alternate position during propagation of
recombinant influenza viruses.
[0104] a) Construction of Bicistronic Expression Plasmid DNAs:
[0105] Starting out with vector plasmid pHH10 (Hoffmann, Ph.D.
Thesis, Univ. Giessen (1997)), i.e. an ampicillin resistant plasmid
including in between a human rDNA promoter segment and a murine
rDNA terminator segment precisely inserted cDNA sequence elements
representing the 5' and 3' vRNA sequence of influenza rRNA segment
5, and finally a central multiple cloning site sequence as obtained
from plasmid PBSK, both reporter genes have been inserted in a
stepwise manner. After that, to the proximal reading frame, i.e.
CAT in pHL3196, and GFP in pHL3224, has been added an upstream
splice donor sequence element and a downstream splice acceptor
element, both inserted as double-strand oligonucleotides, in
between particular restriction cleavage sites available in the
respective positions. The signal sequences used in that pair of
plasmids indicated above have been derived from influenza vRNA
segment 7, which is known for its partial splice reactions yielding
both gene products, M1 and M2, simultaneously. By insertion of a
non-transcribed DNA fragment (representing an internal segment of
the influenza PB1 coding region) in a distal position relative to
both reading frames, pHL3196 has been converted into pHL3235, and
pHL3224 into pHL3236. For the resulting plasmid constructs see
FIGS. 5-8 and SEQ ID NOs: 21-24.
[0106] b) Transfection of Plasmid DNAs and Isolation of Recombinant
Influenza Viruses:
[0107] Semi-confluent 293-T cells, a human renal cacinoma cell line
carrying an artificially integrated tumor virus SV40 T-antigen
gene, were DNA-transfected using lipofectamine: 5-10 .mu.g of DNA
mixed with 30 .mu.l Lipofectamine.RTM. (GIBCO/BRL) were added to
370 .mu.l of DMEM medium and were incubated with 5.times.10.sup.6
to 10.sup.7 cells, washed and maintained serum-free for 5 to 8
hours, before serum was added for another 12 to 15 hours. Finally
influenza helper virus FPV.sub.Bratislava was used for infection of
the DNA-transfected cells. The supernatant containing a mixture of
helper viruses and recombinant viruses was collected for further
propagation after 8 to 12 hours of infection, while the sedimented
cells were used for preparation of a cell lysate, fractions of
which were inserted in the CAT assay procedure.
[0108] Viral propagation was achieved by infection of MDCK cells
(Madin-Darby canine kidney cell line) again in semi-confluent state
(5.times.10.sup.6 to 10.sup.7 cells per plate), generally using 1
ml of the previous supernatant for infection. Serial propagations
were done in the same way, with preparation of cell lysates for CAT
assays at the end of each step. Infected cells were also used for
observation of GFP fluorescence.
[0109] c) CAT Assay:
[0110] Bacterial chloramphenicol-acetyltransferase (CAT) is
accumulated in eukaryotic cells without degradation and can be used
for representative gene expression measurements. The substrate used
here is fluorescent boron-dipyrromethane-chloramphenicol diflouride
(FLASH CAT-KIT.RTM.; Stratagene). 50 .mu.l of cell lysate or
reduced/diluted samples thereof were used for incubation with 7.5
.mu.l of fluorescent substrate and 10 .mu.l acetyl-CoA (4 mM)
co-substrate in 19 mM Tris/HCl, pH: 7.5 at 37.degree. C. for 3
hours. For extraction of reaction products 1 ml of ethylacetate ins
added, the mixture is vortexed, and separated by centrifugation.
After solvent evaporation and dissolution again in 20 ml
ethylacetate, the reaction products are separated on a silica
thin-layer chramatography plate using chloroform/methanol 87:13%
(vol.) and the results are documented by photography under UV
light.
[0111] d) Results
[0112] CAT in proximal or in distal position of this pair of
recombinant plasmids is expressed about equally (FIG. 4), and the
same is true for GFP (not shown). The expression rates are
increasing during the initial steps of viral propagation and stay
about constant afterwards during further steps of recombinant viral
passages, different from expression rates in ambisense bicistronic
constructs (pHL2899 and pHL2960) (FIG. 4B). Co-transfection of
booster plasmids in the initial 293-T cells increase the yields of
recombinant viruses within the progeny population, which are
maintained during consecutive steps of propagation. Addition of
1000 nucleotides of untranslated vRNA sequence will not reduce the
expression rates substantially (pHL3235 versus pHL3196, and pHL3236
versus pHL3224).
Sequence CWU 1
1
24 1 12 RNA Influenza A virus 1 ccugcuuuug cu 12 2 12 RNA Influenza
B virus misc_feature (1)...(2) n=any nucleotide 2 nnygcuucug cu 12
3 12 RNA Influenza C virus 3 ccugcuucug cu 12 4 12 RNA Artificial
Sequence Description of Artificial Sequence Modified influenza A
3'-sequence (pHL1104 and pHL1920) 4 ccuguuucua cu 12 5 12 RNA
Artificial Sequence Description of Artificial Sequence Modified
influenza A 3'-sequence (pHL1948) 5 ccugguucuc cu 12 6 13 RNA
Influenza A virus 6 aguagaaaca agg 13 7 13 RNA Influenza B virus
misc_feature (12)..(13) n=any nucleotide 7 aguagwaaca rnn 13 8 13
RNA Influenza C virus 8 agcaguagca agr 13 9 13 RNA Artificial
Sequence Description of Artificial Sequence Modified influenza A
5'-sequence (pHL1920) 9 agaagaauca agg 13 10 21 RNA Influenza A
virus misc_feature (14)..(16) n=any nucleotide 10 aguagaaaca
aggnnnuuuu u 21 11 21 RNA Artificial Sequence misc_feature
(14)..(16) n=any nucleotide 11 agaagaauca aggnnnuuuu u 21 12 21 RNA
Influenza B virus misc_feature (12)..(16) n=any nucleotide 12
aguagwaaca rnnnnnuuuu u 21 13 19 RNA Artificial Sequence
Description of Artificial Sequence Modified influenza C 5'-sequence
13 aguaguaaca agrguuuuu 19 14 15 RNA Artificial Sequence
Description of Artificial Sequence Modified influenza A 3'-sequence
(pHL1104 and pHL1920) 14 nnnccuguuu cuacu 15 15 15 RNA Artificial
Sequence misc_feature (1)...(3) n=any nucleotide 15 nnnccugguu
cuccu 15 16 15 RNA Artificial Sequence misc_feature (1)...(5) n=any
nucleotide 16 nnnnnyguuu cuacu 15 17 14 RNA Artificial Sequence
Description of Artificial Sequence Modified influenza C 3'-sequence
17 ccccuguuuc uacu 14 18 10 DNA Influenza A virus 18 aggtacgttc 10
19 32 DNA Influenza A virus 19 gctgaaaaat gatcttcttg aaaattgcag gc
32 20 3888 DNA Artificial Sequence Description of Artificial
Sequence pHL1920 20 cccaaaaaaa aaaaaaaaaa aaaaaaaaag agtccagagt
ggccccgccg ttccgcgccg 60 gggggggggg ggggggggga cactttcgga
catctggtcg acctccagca tcgggggaaa 120 aaaaaaaaac aaagtttcgc
ccggagtact ggtcgacctc cgaagttggg ggggagtaga 180 aacagggtag
ataatcactc actgagtgac atccacatcg cgagcgcgcg taatacgact 240
cactataggg cgaattgggt accgggcccc ccctcgaggt cgacggtatc gataagcttc
300 gacgagattt tcaggagcta aggaagctaa aatggagaaa aaaatcactg
gatataccac 360 cgttgatata tcccaatggc atcgtaaaga acattttgag
gcatttcagt cagttgctca 420 atgtacctat aaccagaccg ttcagctgga
tattacggcc tttttaaaga ccgtaaagaa 480 aaataagcac aagttttatc
cggcctttat tcacattctt gcccgcctga tgaatgctca 540 tccggaattc
cgtatggcaa tgaaagacgg tgagctggtg atatgggata gtgttcaccc 600
ttgttacacc gttttccatg agcaaactga aacgttttca tcgctctgga gtgaatacca
660 cgacgatttc cggcagtttc tacacatata ttcgcaagat gtggcgtgtt
acggtgaaaa 720 cctggcctat ttccctaaag ggtttattga gaatatgttt
ttcgtctcag ccaatccctg 780 ggtgagtttc accagttttg atttaaacgt
ggccaatatg gacaacttct tcgcccccgt 840 tttcaccatg ggcaaatatt
atacgcaagg cgacaaggtg ctgatgccgc tggcgattca 900 ggttcatcat
gccgtttgtg atggcttcca tgtcggcaga atgcttaatg aattacaaca 960
gtactgcgat gagtggcagg gcggggcgta atttttttaa ggcagttatt ggtgccctta
1020 aacgcctggt gctacgcctg aataagtgat aataagcgga tgaatggcag
aaattcgtcg 1080 aagcttgata tcgaattcct gcagcccggg ggatccacta
gttctagagc ggccgccacc 1140 gcggtggagc tccagctttt gttcccttta
gtgagggtta attgcgcgca ggcctagcta 1200 ggtaaagaaa aatacccttg
attcttctaa taacccggcg gcccaaaatg ccgactcgga 1260 gcgaaagata
tacctccccc ggggccggga ggtcgcgtca ccgaccacgc cgccggccca 1320
ggcgacgcgc gacacggaca cctgtcccca aaaacgccac catcgcagcc acacacggag
1380 cgcccggggc cctctggtca accccaggac acacgcggga gcagcgccgg
gccggggacg 1440 ccctcccggc cgcccgtgcc acacgcaggg ggccggcccg
tgtctccaga gcgggagccg 1500 gaagcatttt cggccggccc ctcctacgac
cgggacacac gagggaccga aggccggcca 1560 ggcgcgacct ctcgggccgc
acgcgcgctc agggagcgct ctccgactcc gcacggggac 1620 tcgccagaaa
ggatcgtgac ctgcattaat gaatcagggg ataacgcagg aaagaacatg 1680
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
1740 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
gaggtggcga 1800 aacccgacag gactataaag ataccaggcg tttccccctg
gaagctccct cgtgcgctct 1860 cctgttccga ccctgccgct taccggatac
ctgtccgcct ttctcccttc gggaagcgtg 1920 gcgctttctc atagctcacg
ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 1980 ctgggctgtg
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 2040
cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
2100 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
gtggcctaac 2160 tacggctaca ctagaaggac agtatttggt atctgcgctc
tgctgaagcc agttaccttc 2220 ggaaaaagag ttggtagctc ttgatccggc
aaacaaacca ccgctggtag cggtggtttt 2280 tttgtttgca agcagcagat
tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 2340 ttttctacgg
ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 2400
agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
2460 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
cagtgaggca 2520 cctatctcag cgatctgtct atttcgttca tccatagttg
cctgactccc cgtcgtgtag 2580 ataactacga tacgggaggg cttaccatct
ggccccagtg ctgcaatgat accgcgagac 2640 ccacgctcac cggctccaga
tttatcagca ataaaccagc cagccggaag ggccgagcgc 2700 agaagtggtc
ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 2760
agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
2820 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
acgatcaagg 2880 cgagttacat gatcccccat gttgtgcaaa aaagcggtta
gctccttcgg tcctccgatc 2940 gttgtcagaa gtaagttggc cgcagtgtta
tcactcatgg ttatggcagc actgcataat 3000 tctcttactg tcatgccatc
cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 3060 tcattctgag
aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat 3120
aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
3180 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
cactcgtgca 3240 cccaactgat cttcagcatc ttttactttc accagcgttt
ctgggtgagc aaaaacagga 3300 aggcaaaatg ccgcaaaaaa gggaataagg
gcgacacgga aatgttgaat actcatactc 3360 ttcctttttc aatattattg
aagcatttat cagggttatt gtctcatgag cggatacata 3420 tttgaatgta
tttagaaaaa taaacaaaag agtttgtaga aacgcaaaaa ggccatccgt 3480
caggatggcc ttctgcttaa tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca
3540 ccctccgggc cgttgcttcg caacgttcaa atccgctccc ggcggatttg
tcctactcag 3600 gagagcgttc accgacaaac aacagataaa acgaaaggcc
cagtctttcg actgagcctt 3660 tcgttttatt tgatgcctgg cagttcccta
ctctcgcatg gggagacccc acactaccat 3720 cggcgctacg gcgtttcact
tctgagttcg gcatggggtc aggtgggacc accgcgctac 3780 tgccgccagg
caaattctgt tttatcagac cgcttctgcg ttctgattta atctgtatca 3840
ggctgaaaat cttctctcat ccgccaaaac agaagctagc ggccgatc 3888 21 4500
DNA Artificial Sequence Description of Artificial Sequence pHL3196
21 agtagaaaca gggtagataa tcactcactg agtgacatcc acatcgcgag
cgcgaaggta 60 cgttctcgag cgcgcgtaat acgactcact atagggcgaa
ttgggtacgt tccatcatgg 120 agaaaaaaat cactggatat accaccgttg
atatatccca atggcatcgt aaagaacatt 180 ttgaggcatt tcagtcagtt
gctcaatgta cctataacca gaccgttcag ctggatatta 240 cggccttttt
aaagaccgta aagaaaaata agcacaagtt ttatccggcc tttattcaca 300
ttcttgcccg cctgatgaat gctcatccgg aattccgtat ggcaatgaaa gacggtgagc
360 tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa
actgaaacgt 420 tttcatcgct ctggagtgaa taccacgacg atttccggca
gtttctacac atatattcgc 480 aagatgtggc gtgttacggt gaaaacctgg
cctatttccc taaagggttt attgagaata 540 tgtttttcgt ctcagccaat
ccctgggtga gtttcaccag ttttgattta aacgtggcca 600 atatggacaa
cttcttcgcc cccgttttca ccatgggcaa atattatacg caaggcgaca 660
aggtgctgat gccgctggcg attcaggttc atcatgccgt ctgtgatggc ttccatgtcg
720 gcagaatgct taatgaatta caacagtact gcgatgagtg gcagggcggg
gcgcgttaac 780 gagatcagct gaaaaatgat cttcttgaaa atttgcaggc
cgtacgtgta ccgggccccc 840 cctcgactcg cgaaggagtc caccatgagt
aaaggagaag aacttttcac tggagttgtc 900 ccaattcttg ttgaattaga
tggtgatgtt aatgggcaca aattttctgt cagtggagag 960 ggtgaaggtg
atgcaacata cggaaaactt acccttaaat ttatttgcac tactggaaaa 1020
ctacctgttc catggccaac acttgtcact actttcactt atggtgttca atgcttttca
1080 agatacccag atcatatgaa acagcatgac tttttcaaga gtgccatgcc
cgaaggttat 1140 gtacaggaaa gaactatatt tttcaaagat gacgggaact
acaagacacg tgctgaagtc 1200 aagtttgaag gtgataccct tgttaataga
atcgagttaa aaggtattga ttttaaagaa 1260 gatggaaaca ttcttggaca
caaattggaa tacaactata actcacacaa tgtatacatc 1320 atggctgaca
agcagaagaa cggaatcaag gccaacttca agacccgcca caacatcgag 1380
gacggcggcg tgcagctggc cgaccactac cagcagaaca ccccaattgg cgatggccct
1440 gtccttttac cagacaacca ttacctgtcc acacaatctg ccctttcgaa
agatcccaac 1500 gaaaagagag accacatggt ccttcttgag tttgtaacag
ctgctgggat tacacatggc 1560 atggatgaac tatacaaggg atcccatcac
catcaccatc actaagctcc atggtctaga 1620 tatcgatagg cctagctagg
taaagaaaaa tacccttgtt tctactaata acccggcggc 1680 ccaaaatgcc
gactcggagc gaaagatata cctcccccgg ggccgggagg tcgcgtcacc 1740
gaccacgccg ccggcccagg cgacgcgcga cacggacacc tgtccccaaa aacgccacca
1800 tcgcagccac acacggagcg cccggggccc tctggtcaac cccaggacac
acgcgggagc 1860 agcgccgggc cggggacgcc ctcccggccg cccgtgccac
acgcaggggg ccggcccgtg 1920 tctccagagc gggagccgga agcattttcg
gccggcccct cctacgaccg ggacacacga 1980 gggaccgaag gccggccagg
cgcgacctct cgggccgcac gcgcgctcag ggagcgctct 2040 ccgactccgc
acggggactc gccagaaagg atcgtgacct gcattaatga atcaggggat 2100
aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
2160 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
aaatcgacgc 2220 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
accaggcgtt tccccctgga 2280 agctccctcg tgcgctctcc tgttccgacc
ctgccgctta ccggatacct gtccgccttt 2340 ctcccttcgg gaagcgtggc
gctttctcat agctcacgct gtaggtatct cagttcggtg 2400 taggtcgttc
gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 2460
gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
2520 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
tacagagttc 2580 ttgaagtggt ggcctaacta cggctacact agaaggacag
tatttggtat ctgcgctctg 2640 ctgaagccag ttaccttcgg aaaaagagtt
ggtagctctt gatccggcaa acaaaccacc 2700 gctggtagcg gtggtttttt
tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 2760 caagaagatc
ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 2820
taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
2880 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
cagttaccaa 2940 tgcttaatca gtgaggcacc tatctcagcg atctgtctat
ttcgttcatc catagttgcc 3000 tgactccccg tcgtgtagat aactacgata
cgggagggct taccatctgg ccccagtgct 3060 gcaatgatac cgcgagaccc
acgctcaccg gctccagatt tatcagcaat aaaccagcca 3120 gccggaaggg
ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 3180
aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
3240 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
attcagctcc 3300 ggttcccaac gatcaaggcg agttacatga tcccccatgt
tgtgcaaaaa agcggttagc 3360 tccttcggtc ctccgatcgt tgtcagaagt
aagttggccg cagtgttatc actcatggtt 3420 atggcagcac tgcataattc
tcttactgtc atgccatccg taagatgctt ttctgtgact 3480 ggtgagtact
caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 3540
ccggcgtcaa cacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
3600 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
atccagttcg 3660 atgtaaccca ctcgtgcacc caactgatct tcagcatctt
ttactttcac cagcgtttct 3720 gggtgagcaa aaacaggaag gcaaaatgcc
gcaaaaaagg gaataagggc gacacggaaa 3780 tgttgaatac tcatactctt
cctttttcaa tattattgaa gcatttatca gggttattgt 3840 ctcatgagcg
gatacatatt tgaatgtatt tagaaaaata aacaaaagag tttgtagaaa 3900
cgcaaaaagg ccatccgtca ggatggcctt ctgcttaatt tgatgcctgg cagtttatgg
3960 cgggcgtcct gcccgccacc ctccgggccg ttgcttcgca acgttcaaat
ccgctcccgg 4020 cggatttgtc ctactcagga gagcgttcac cgacaaacaa
cagataaaac gaaaggccca 4080 gtctttcgac tgagcctttc gttttatttg
atgcctggca gttccctact ctcgcatggg 4140 gagaccccac actaccatcg
gcgctacggc gtttcacttc tgagttcggc atggggtcag 4200 gtgggaccac
cgcgctactg ccgccaggca aattctgttt tatcagaccg cttctgcgtt 4260
ctgatttaat ctgtatcagg ctgaaaatct tctctcatcc gccaaaacag aagctagcgg
4320 ccgatcccca aaaaaaaaaa aaaaaaaaaa aaaaagagtc cagagtggcc
ccgccgttcc 4380 gcgccggggg gggggggggg gggggacact ttcggacatc
tggtcgacct ccagcatcgg 4440 gggaaaaaaa aaaaacaaag tttcgcccgg
agtactggtc gacctccgaa gttggggggg 4500 22 4721 DNA Artificial
Sequence Description of Artificial Sequence pHL3224 22 atctagacca
tggagcttag tgatggtgat ggtgatggga tcccttgtat agttcatcca 60
tgccatgtgt aatcccagca gctgttacaa actcaagaag gaccatgtgg tctctctttt
120 cgttgggatc tttcgaaagg gcagattgtg tggacaggta atggttgtct
ggtaaaagga 180 cagggccatc gccaattggg gtgttctgct ggtagtggtc
ggccagctgc acgccgccgt 240 cctcgatgtt gtggcgggtc ttgaagttgg
ccttgattcc gttcttctgc ttgtcagcca 300 tgatgtatac attgtgtgag
ttatagttgt attccaattt gtgtccaaga atgtttccat 360 cttctttaaa
atcaatacct tttaactcga ttctattaac aagggtatca ccttcaaact 420
tgacttcagc acgtgtcttg tagttcccgt catctttgaa aaatatagtt ctttcctgta
480 cataaccttc gggcatggca ctcttgaaaa agtcatgctg tttcatatga
tctgggtatc 540 ttgaaaagca ttgaacacca taagtgaaag tagtgacaag
tgttggccat ggaacaggta 600 gttttccagt agtgcaaata aatttaaggg
taagttttcc gtatgttgca tcaccttcac 660 cctctccact gacagaaaat
ttgtgcccat taacatcacc atctaattca acaagaattg 720 ggacaactcc
agtgaaaagt tcttctcctt tactcatggt ggactccttc gcgagtcgag 780
ggggggcccg gtacacgtac gcgctcgaga acgtaccttc gcgctcgcga tgtggatgtc
840 actcagtgag tgattatcta ccctgtttct actccccccc aacttcggag
gtcgaccagt 900 actccgggcg aaactttgtt tttttttttt cccccgatgc
tggaggtcga ccagatgtcc 960 gaaagtgtcc cccccccccc ccccccccgg
cgcggaacgg cggggccact ctggactctt 1020 tttttttttt tttttttttt
ttttggggat cggccgctag cttctgtttt ggcggatgag 1080 agaagatttt
cagcctgata cagattaaat cagaacgcag aagcggtctg ataaaacaga 1140
atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac tcagaagtga
1200 aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg
aactgccagg 1260 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct
ttcgttttat ctgttgtttg 1320 tcggtgaacg ctctcctgag taggacaaat
ccgccgggag cggatttgaa cgttgcgaag 1380 caacggcccg gagggtggcg
ggcaggacgc ccgccataaa ctgccaggca tcaaattaag 1440 cagaaggcca
tcctgacgga tggccttttt gcgtttctac aaactctttt gtttattttt 1500
ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
1560 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
ttcccttttt 1620 tgcggcattt tgccttcctg tttttgctca cccagaaacg
ctggtgaaag taaaagatgc 1680 tgaagatcag ttgggtgcac gagtgggtta
catcgaactg gatctcaaca gcggtaagat 1740 ccttgagagt tttcgccccg
aagaacgttt tccaatgatg agcactttta aagttctgct 1800 atgtggcgcg
gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca 1860
ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
1920 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
ctgcggccaa 1980 cttacttctg acaacgatcg gaggaccgaa ggagctaacc
gcttttttgc acaacatggg 2040 ggatcatgta actcgccttg atcgttggga
accggagctg aatgaagcca taccaaacga 2100 cgagcgtgac accacgatgc
ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg 2160 cgaactactt
actctagctt cccggcaaca attaatagac tggatggagg cggataaagt 2220
tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
2280 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
gtaagccctc 2340 ccgtatcgta gttatctaca cgacggggag tcaggcaact
atggatgaac gaaatagaca 2400 gatcgctgag ataggtgcct cactgattaa
gcattggtaa ctgtcagacc aagtttactc 2460 atatatactt tagattgatt
taaaacttca tttttaattt aaaaggatct aggtgaagat 2520 cctttttgat
aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 2580
agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
2640 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
atcaagagct 2700 accaactctt tttccgaagg taactggctt cagcagagcg
cagataccaa atactgtcct 2760 tctagtgtag ccgtagttag gccaccactt
caagaactct gtagcaccgc ctacatacct 2820 cgctctgcta atcctgttac
cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 2880 gttggactca
agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 2940
gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
3000 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
cggtaagcgg 3060 cagggtcgga acaggagagc gcacgaggga gcttccaggg
ggaaacgcct ggtatcttta 3120 tagtcctgtc gggtttcgcc acctctgact
tgagcgtcga tttttgtgat gctcgtcagg 3180 ggggcggagc ctatggaaaa
acgccagcaa cgcggccttt ttacggttcc tggccttttg 3240 ctggcctttt
gctcacatgt tctttcctgc gttatcccct gattcattaa tgcaggtcac 3300
gatcctttct ggcgagtccc cgtgcggagt cggagagcgc tccctgagcg cgcgtgcggc
3360 ccgagaggtc gcgcctggcc ggccttcggt ccctcgtgtg tcccggtcgt
aggaggggcc 3420 ggccgaaaat gcttccggct cccgctctgg agacacgggc
cggccccctg cgtgtggcac 3480 gggcggccgg gagggcgtcc ccggcccggc
gctgctcccg cgtgtgtcct ggggttgacc 3540 agagggcccc gggcgctccg
tgtgtggctg cgatggtggc gtttttgggg acaggtgtcc 3600 gtgtcgcgcg
tcgcctgggc cggcggcgtg gtcggtgacg cgacctcccg gccccggggg 3660
aggtatatct ttcgctccga gtcggcattt tgggccgccg ggttattagt agaaacaagg
3720 gtatttttct ttacctagct aggcctgcgc gcaattaacc ctcactaaag
ggaacaaaag 3780 ctggagctcc accgcggtgg cggccgctct agaactagtg
gatcccccgg gctgcaggaa 3840 ttcgatatca agcttcgacg aatttctgcc
attcatccgc ttattatcac ttattcaggc 3900 gtagcaccag gcgtttaagg
gcaccaataa ctgccttaaa aaaattacgc cccgccctgc 3960 cactcatcgc
agtactgttg taattcatta agcattctgc cgacatggaa gccatcacaa 4020
acggcatgat gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtataatat
4080 ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt
taaatcaaaa 4140
ctggtgaaac tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta
4200 gggaaatagg ccaggttttc accgtaacac gccacatctt gcgaatatat
gtgtagaaac 4260 tgccggaaat cgtcgtggta ttcactccag agcgatgaaa
acgtttcagt ttgctcatgg 4320 aaaacggtgt aacaagggtg aacactatcc
catatcacca gctcaccgtc tttcattgcc 4380 atacggaatt ccggatgagc
attcatcagg cgggcaagaa tgtgaataaa ggccggataa 4440 aacttgtgct
tatttttctt tacggtcttt aaaaaggccg taatatccag ctgaacggtc 4500
tggttatagg tacattgagc aactgactga aatgcctcaa aatgttcttt acgatgccat
4560 tgggatatat caacggtggt atatccagtg atttttttct ccattttagc
ttccttagct 4620 cctgaaaatc tcgtcgaagc ttatcgatac cgtcgacctc
gagggggggc ccggtacggc 4680 ctgcaaattt tcaagaagat catttttcag
ctgatctcgt t 4721 23 5517 DNA Artificial Sequence Description of
Artificial Sequence pHL3235 23 agtagaaaca gggtagataa tcactcactg
agtgacatcc acatcgcgag cgcgaaggta 60 cgttctcgag cgcgcgtaat
acgactcact atagggcgaa ttgggtacgt tccatcatgg 120 agaaaaaaat
cactggatat accaccgttg atatatccca atggcatcgt aaagaacatt 180
ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag ctggatatta
240 cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc
tttattcaca 300 ttcttgcccg cctgatgaat gctcatccgg aattccgtat
ggcaatgaaa gacggtgagc 360 tggtgatatg ggatagtgtt cacccttgtt
acaccgtttt ccatgagcaa actgaaacgt 420 tttcatcgct ctggagtgaa
taccacgacg atttccggca gtttctacac atatattcgc 480 aagatgtggc
gtgttacggt gaaaacctgg cctatttccc taaagggttt attgagaata 540
tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta aacgtggcca
600 atatggacaa cttcttcgcc cccgttttca ccatgggcaa atattatacg
caaggcgaca 660 aggtgctgat gccgctggcg attcaggttc atcatgccgt
ctgtgatggc ttccatgtcg 720 gcagaatgct taatgaatta caacagtact
gcgatgagtg gcagggcggg gcgcgttaac 780 gagatcagct gaaaaatgat
cttcttgaaa atttgcaggc cgtacgtgta ccgggccccc 840 cctcgactcg
cgaaggagtc caccatgagt aaaggagaag aacttttcac tggagttgtc 900
ccaattcttg ttgaattaga tggtgatgtt aatgggcaca aattttctgt cagtggagag
960 ggtgaaggtg atgcaacata cggaaaactt acccttaaat ttatttgcac
tactggaaaa 1020 ctacctgttc catggccaac acttgtcact actttcactt
atggtgttca atgcttttca 1080 agatacccag atcatatgaa acagcatgac
tttttcaaga gtgccatgcc cgaaggttat 1140 gtacaggaaa gaactatatt
tttcaaagat gacgggaact acaagacacg tgctgaagtc 1200 aagtttgaag
gtgataccct tgttaataga atcgagttaa aaggtattga ttttaaagaa 1260
gatggaaaca ttcttggaca caaattggaa tacaactata actcacacaa tgtatacatc
1320 atggctgaca agcagaagaa cggaatcaag gccaacttca agacccgcca
caacatcgag 1380 gacggcggcg tgcagctggc cgaccactac cagcagaaca
ccccaattgg cgatggccct 1440 gtccttttac cagacaacca ttacctgtcc
acacaatctg ccctttcgaa agatcccaac 1500 gaaaagagag accacatggt
ccttcttgag tttgtaacag ctgctgggat tacacatggc 1560 atggatgaac
tatacaaggg atcttcatga tctcagcaaa ctcttccttc ttaatccttc 1620
cagactcgaa gtcaattcgt gcatcaatcc gggccctaga caccatggcc tccaccatac
1680 tggaaattcc aactggtctt ctgtatgagc tgctagggaa gaatttctcg
aataggttgc 1740 aacacttctg gtacatttgt tcatcctcaa ggattcccct
ttgactcgta ttgagaatgg 1800 aacggtttct cttagggatc caagagtgtg
tagttgccac agcatcatat tccatgcttt 1860 tggctggacc atgggctggc
attaccgcag cattgtttac agattcaatt tccttatgac 1920 tgacaaacgg
gttcatggga ttacaaagtc ttccctgata gtcttcatcc attagttccc 1980
atttcaggca aacttccggg atgtggagat tccgaatgtt gtacaggttt ggtccgccat
2040 ctgaaaccaa cagtcctgcc tttgagcggg tctgctccca cagcttcttt
agctcgaatg 2100 acctcctcgt ttggatttgt gtgtctcccc tgtgacaccg
gtatgtatat ctgtagtcct 2160 tgatgaataa ttggagagcc atttgggctg
ttgccggtcc aagatcattg tttatcatgt 2220 tattctttat cactgttact
ccaatgctca tatcagccga ttcattaatt cctgatactc 2280 caaagctggg
caactccata ctaaaattgg ctacaaatcc atagcggtag aaaaagcttg 2340
tgaattcgaa tgttcctgtc ctatttatat aggacttttt cttgctcata ttgatcccaa
2400 ctagcttgca ggttctgtag aatctatcca ctcccgcttg tattccctca
tgatttggtg 2460 cattcacgat gagagcaaaa tcatcagagg actgaagtcc
atcccaccag tatgtggttt 2520 tggtgtatct cttttgccca agattcagga
ttgagactcc caacactgta ctcagcatgt 2580 tgaacatacc catcatcatt
cccgggctta atgaggctgt gccgtctatt atgagaggat 2640 cgataggcct
agctaggtaa agaaaaatac ccttgtttct actaataacc cggcggccca 2700
aaatgccgac tcggagcgaa agatatacct cccccggggc cgggaggtcg cgtcaccgac
2760 cacgccgccg gcccaggcga cgcgcgacac ggacacctgt ccccaaaaac
gccaccatcg 2820 cagccacaca cggagcgccc ggggccctct ggtcaacccc
aggacacacg cgggagcagc 2880 gccgggccgg ggacgccctc ccggccgccc
gtgccacacg cagggggccg gcccgtgtct 2940 ccagagcggg agccggaagc
attttcggcc ggcccctcct acgaccggga cacacgaggg 3000 accgaaggcc
ggccaggcgc gacctctcgg gccgcacgcg cgctcaggga gcgctctccg 3060
actccgcacg gggactcgcc agaaaggatc gtgacctgca ttaatgaatc aggggataac
3120 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
aaaggccgcg 3180 ttgctggcgt ttttccatag gctccgcccc cctgacgagc
atcacaaaaa tcgacgctca 3240 agtcagaggt ggcgaaaccc gacaggacta
taaagatacc aggcgtttcc ccctggaagc 3300 tccctcgtgc gctctcctgt
tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 3360 ccttcgggaa
gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 3420
gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
3480 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
gccactggca 3540 gcagccactg gtaacaggat tagcagagcg aggtatgtag
gcggtgctac agagttcttg 3600 aagtggtggc ctaactacgg ctacactaga
aggacagtat ttggtatctg cgctctgctg 3660 aagccagtta ccttcggaaa
aagagttggt agctcttgat ccggcaaaca aaccaccgct 3720 ggtagcggtg
gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 3780
gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
3840 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
aaattaaaaa 3900 tgaagtttta aatcaatcta aagtatatat gagtaaactt
ggtctgacag ttaccaatgc 3960 ttaatcagtg aggcacctat ctcagcgatc
tgtctatttc gttcatccat agttgcctga 4020 ctccccgtcg tgtagataac
tacgatacgg gagggcttac catctggccc cagtgctgca 4080 atgataccgc
gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 4140
ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
4200 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
cgttgttgcc 4260 attgctacag gcatcgtggt gtcacgctcg tcgtttggta
tggcttcatt cagctccggt 4320 tcccaacgat caaggcgagt tacatgatcc
cccatgttgt gcaaaaaagc ggttagctcc 4380 ttcggtcctc cgatcgttgt
cagaagtaag ttggccgcag tgttatcact catggttatg 4440 gcagcactgc
ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 4500
gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
4560 gcgtcaacac gggataatac cgcgccacat agcagaactt taaaagtgct
catcattgga 4620 aaacgttctt cggggcgaaa actctcaagg atcttaccgc
tgttgagatc cagttcgatg 4680 taacccactc gtgcacccaa ctgatcttca
gcatctttta ctttcaccag cgtttctggg 4740 tgagcaaaaa caggaaggca
aaatgccgca aaaaagggaa taagggcgac acggaaatgt 4800 tgaatactca
tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 4860
atgagcggat acatatttga atgtatttag aaaaataaac aaaagagttt gtagaaacgc
4920 aaaaaggcca tccgtcagga tggccttctg cttaatttga tgcctggcag
tttatggcgg 4980 gcgtcctgcc cgccaccctc cgggccgttg cttcgcaacg
ttcaaatccg ctcccggcgg 5040 atttgtccta ctcaggagag cgttcaccga
caaacaacag ataaaacgaa aggcccagtc 5100 tttcgactga gcctttcgtt
ttatttgatg cctggcagtt ccctactctc gcatggggag 5160 accccacact
accatcggcg ctacggcgtt tcacttctga gttcggcatg gggtcaggtg 5220
ggaccaccgc gctactgccg ccaggcaaat tctgttttat cagaccgctt ctgcgttctg
5280 atttaatctg tatcaggctg aaaatcttct ctcatccgcc aaaacagaag
ctagcggccg 5340 atccccaaaa aaaaaaaaaa aaaaaaaaaa aagagtccag
agtggccccg ccgttccgcg 5400 ccgggggggg gggggggggg ggacactttc
ggacatctgg tcgacctcca gcatcggggg 5460 aaaaaaaaaa aacaaagttt
cgcccggagt actggtcgac ctccgaagtt ggggggg 5517 24 5699 DNA
Artificial Sequence Description of Artificial Sequence pHL3236 24
cctctcataa tagacggcac agcctcatta agcccgggaa tgatgatggg tatgttcaac
60 atgctgagta cagtgttggg agtctcaatc ctgaatcttg ggcaaaagag
atacaccaaa 120 accacatact ggtgggatgg acttcagtcc tctgatgatt
ttgctctcat cgtgaatgca 180 ccaaatcatg agggaataca agcgggagtg
gatagattct acagaacctg caagctagtt 240 gggatcaata tgagcaagaa
aaagtcctat ataaatagga caggaacatt cgaattcaca 300 agctttttct
accgctatgg atttgtagcc aattttagta tggagttgcc cagctttgga 360
gtatcaggaa ttaatgaatc ggctgatatg agcattggag taacagtgat aaagaataac
420 atgataaaca atgatcttgg accggcaaca gcccaaatgg ctctccaatt
attcatcaag 480 gactacagat atacataccg gtgtcacagg ggagacacac
aaatccaaac gaggaggtca 540 ttcgagctaa agaagctgtg ggagcagacc
cgctcaaagg caggactgtt ggtttcagat 600 ggcggaccaa acctgtacaa
cattcggaat ctccacatcc cggaagtttg cctgaaatgg 660 gaactaatgg
atgaagacta tcagggaaga ctttgtaatc ccatgaaccc gtttgtcagt 720
cataaggaaa ttgaatctgt aaacaatgct gcggtaatgc cagcccatgg tccagccaaa
780 agcatggaat atgatgctgt ggcaactaca cactcttgga tccctaagag
aaaccgttcc 840 attctcaata cgagtcaaag gggaatcctt gaggatgaac
aaatgtacca gaagtgttgc 900 aacctattcg agaaattctt ccctagcagc
tcatacagaa gaccagttgg aatttccagt 960 atggtggagg ccatggtgtc
tagggcccgg attgatgcac gaattgactt cgagtctgga 1020 aggattaaga
aggaagagtt tgctgagatc atgaagatcc cccgggctgc aggaattcga 1080
tatcaagctt cgacgaattt ctgccattca tccgcttatt atcacttatt caggcgtagc
1140 accaggcgtt taagggcacc aataactgcc ttaaaaaaat tacgccccgc
cctgccactc 1200 atcgcagtac tgttgtaatt cattaagcat tctgccgaca
tggaagccat cacaaacggc 1260 atgatgaacc tgaatcgcca gcggcatcag
caccttgtcg ccttgcgtat aatatttgcc 1320 catggtgaaa acgggggcga
agaagttgtc catattggcc acgtttaaat caaaactggt 1380 gaaactcacc
cagggattgg ctgagacgaa aaacatattc tcaataaacc ctttagggaa 1440
ataggccagg ttttcaccgt aacacgccac atcttgcgaa tatatgtgta gaaactgccg
1500 gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct
catggaaaac 1560 ggtgtaacaa gggtgaacac tatcccatat caccagctca
ccgtctttca ttgccatacg 1620 gaattccgga tgagcattca tcaggcgggc
aagaatgtga ataaaggccg gataaaactt 1680 gtgcttattt ttctttacgg
tctttaaaaa ggccgtaata tccagctgaa cggtctggtt 1740 ataggtacat
tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga 1800
tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga
1860 aaatctcgtc gaagcttatc gataccgtcg acctcgaggg ggggcccggt
acggcctgca 1920 aattttcaag aagatcattt ttcagctgat ctcgttatct
agaccatgga gcttagtgat 1980 ggtgatggtg atgggatccc ttgtatagtt
catccatgcc atgtgtaatc ccagcagctg 2040 ttacaaactc aagaaggacc
atgtggtctc tcttttcgtt gggatctttc gaaagggcag 2100 attgtgtgga
caggtaatgg ttgtctggta aaaggacagg gccatcgcca attggggtgt 2160
tctgctggta gtggtcggcc agctgcacgc cgccgtcctc gatgttgtgg cgggtcttga
2220 agttggcctt gattccgttc ttctgcttgt cagccatgat gtatacattg
tgtgagttat 2280 agttgtattc caatttgtgt ccaagaatgt ttccatcttc
tttaaaatca atacctttta 2340 actcgattct attaacaagg gtatcacctt
caaacttgac ttcagcacgt gtcttgtagt 2400 tcccgtcatc tttgaaaaat
atagttcttt cctgtacata accttcgggc atggcactct 2460 tgaaaaagtc
atgctgtttc atatgatctg ggtatcttga aaagcattga acaccataag 2520
tgaaagtagt gacaagtgtt ggccatggaa caggtagttt tccagtagtg caaataaatt
2580 taagggtaag ttttccgtat gttgcatcac cttcaccctc tccactgaca
gaaaatttgt 2640 gcccattaac atcaccatct aattcaacaa gaattgggac
aactccagtg aaaagttctt 2700 ctcctttact catggtggac tccttcgcga
gtcgaggggg ggcccggtac acgtacgcgc 2760 tcgagaacgt accttcgcgc
tcgcgatgtg gatgtcactc agtgagtgat tatctaccct 2820 gtttctactc
ccccccaact tcggaggtcg accagtactc cgggcgaaac tttgtttttt 2880
ttttttcccc cgatgctgga ggtcgaccag atgtccgaaa gtgtcccccc cccccccccc
2940 ccccggcgcg gaacggcggg gccactctgg actctttttt tttttttttt
tttttttttt 3000 ggggatcggc cgctagcttc tgttttggcg gatgagagaa
gattttcagc ctgatacaga 3060 ttaaatcaga acgcagaagc ggtctgataa
aacagaattt gcctggcggc agtagcgcgg 3120 tggtcccacc tgaccccatg
ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg 3180 tggggtctcc
ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag 3240
tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
3300 acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg
gtggcgggca 3360 ggacgcccgc cataaactgc caggcatcaa attaagcaga
aggccatcct gacggatggc 3420 ctttttgcgt ttctacaaac tcttttgttt
atttttctaa atacattcaa atatgtatcc 3480 gctcatgaga caataaccct
gataaatgct tcaataatat tgaaaaagga agagtatgag 3540 tattcaacat
ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 3600
tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
3660 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
gccccgaaga 3720 acgttttcca atgatgagca cttttaaagt tctgctatgt
ggcgcggtat tatcccgtgt 3780 tgacgccggg caagagcaac tcggtcgccg
catacactat tctcagaatg acttggttga 3840 gtactcacca gtcacagaaa
agcatcttac ggatggcatg acagtaagag aattatgcag 3900 tgctgccata
accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 3960
accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
4020 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
cgatgcctgt 4080 agcaatggca acaacgttgc gcaaactatt aactggcgaa
ctacttactc tagcttcccg 4140 gcaacaatta atagactgga tggaggcgga
taaagttgca ggaccacttc tgcgctcggc 4200 ccttccggct ggctggttta
ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 4260 tatcattgca
gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 4320
ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
4380 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
ttgatttaaa 4440 acttcatttt taatttaaaa ggatctaggt gaagatcctt
tttgataatc tcatgaccaa 4500 aatcccttaa cgtgagtttt cgttccactg
agcgtcagac cccgtagaaa agatcaaagg 4560 atcttcttga gatccttttt
ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 4620 gctaccagcg
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 4680
tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
4740 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
tgttaccagt 4800 ggctgctgcc agtggcgata agtcgtgtct taccgggttg
gactcaagac gatagttacc 4860 ggataaggcg cagcggtcgg gctgaacggg
gggttcgtgc acacagccca gcttggagcg 4920 aacgacctac accgaactga
gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 4980 cgaagggaga
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 5040
gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
5100 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
ggaaaaacgc 5160 cagcaacgcg gcctttttac ggttcctggc cttttgctgg
ccttttgctc acatgttctt 5220 tcctgcgtta tcccctgatt cattaatgca
ggtcacgatc ctttctggcg agtccccgtg 5280 cggagtcgga gagcgctccc
tgagcgcgcg tgcggcccga gaggtcgcgc ctggccggcc 5340 ttcggtccct
cgtgtgtccc ggtcgtagga ggggccggcc gaaaatgctt ccggctcccg 5400
ctctggagac acgggccggc cccctgcgtg tggcacgggc ggccgggagg gcgtccccgg
5460 cccggcgctg ctcccgcgtg tgtcctgggg ttgaccagag ggccccgggc
gctccgtgtg 5520 tggctgcgat ggtggcgttt ttggggacag gtgtccgtgt
cgcgcgtcgc ctgggccggc 5580 ggcgtggtcg gtgacgcgac ctcccggccc
cgggggaggt atatctttcg ctccgagtcg 5640 gcattttggg ccgccgggtt
attagtagaa acaagggtat ttttctttac ctagctagg 5699
* * * * *